The random generation of T cell receptor (TCR) sequences ensures a TCR repertoire capable of recognizing a wide variety of pathogens. However, study of the TCR repertoire is hampered by this great diversity. To overcome this issue, we have used a fixed TCR-beta chain model to facilitate experimental study of a polyclonal TCR repertoire. This TCR repertoire develops in a normal thymic and peripheral environment, and is subject to natural T cell selective forces, including potential interactions with self-antigens and foreign antigens from commensal flora on the skin and intestines. In previous studies, this fixed TCR-beta chain model has been useful in characterizing normal naturally arising TCR repertoires and comparing them with autoimmune repertoires. We propose here that detailed knowledge of the TCR repertoire in a fixed TCR- beta chain model will be useful for study of numerous aspects of T cell biology, Thus, the goal of this project is the complete in situ characterization of this polyclonal TCR repertoire at the individual TCR level by extensive sequencing of the variable TCR-alpha chains from T cells isolated based on spatial and phenotypic variables. One particular area of focus will be to understand the TCR specificity of naturally arising memory CD4+ T cells, which presumably arose due to encounter with self or commensal antigens, and may potentially be pathogenic. We believe these data will serve as the foundation of future studies on self-reactive regulatory and non-regulatory T cells, genetic models of autoimmune disease, memory T cells, and T cell trafficking and homeostasis.